Investigation On Carbon Source Induction Mechanism Ofα-Galactosidase And The Multi-enzyme Development

a-galactosidase (a-D-galactoside galctohydrolases, EC3.2.1.22) belong to exo-glycosidase and can been divided into glycoside hydrolase (GH) families4,27,36,57and97. The enzymes are widely distributed in microorganisms, plants and animals and they specially cleave the terminal of α-1â†'6-linked D-galactosyl residues. The researches about the enzymes had been carried out in our country since1970s. Nowadays, a-galactosidase is considered to be one of the most potential enzymes in various fields like the food industry, light industry, feed industry, the pharmaceutical industry et al. In this paper, safe a-galactosidase-producing strain was screened and bred. Carbon source induced mechanism for a-galactosidase production was discussed. And then the cultivation process and medium composition were optimized. On that basis, the multi-enzymes symbolized by a-galactosidase were developed with agricultural by-products. The primary results of this study were summarized below:The high-yielding strain J-08was obtained from various strains conserved in our lab and traditional fermentation bean products by X-a-Gal preliminary screening, liquid fermentation and solid state fermentation secondary screening. The stain was demominated as zju-Y1with a-galactosidase activity at the level of3.88U/mL and57.21U/g dry matter after liquid fermentation and solid state fermentation respectively. It was identified as Aspergillus niger by morphology, BIOLOG biochemical identification,18S rRNA sequence analysis.The effect of a-galactosidase-producing was investigated with various agricultural by-products. Stachyose was speculated as one of the most effective induction factors for a-galactosidase-producing via Aspergillus niger zju-Yl by analyzing the composite of agricultural by-products. The single carbon source experiment had shown that stachyose had the most induction enzyme production effect among monosaccharides& oligosaccharides and guar gum was the most one among polysaccharide.The expression patterns of three cDNAs (ag1A, ag1B and ag1C), encoding different a-galactosidase of Aspergillus niger were investigated with competitive Real Time PCR under different carbon sources cultivation. The sensitivity of inducing with carbon sources on expression level of three genes was ag1C> ag1B> ag1A. The order of carbon sources induction for genes up-regulated effection was stachyose> guar gum> galactose> glucose. The expression patterns regulatory factor of of Aspergillus niger zju-Y1were investigated. It conjectured that activating transcription factor XlnR and AmyR were the regulating factors for the a-galactosidase gene expression.Single-factor experiments was used to screen the preferable fermentation condition.The optimum culture condition is28℃, medium initial moisture60%, initial pH5.5, inoculum size12%, culture load25g (wet matter) with250mL flask.RSM was applied to optimize the composition of medium, the results showed that the optium fermentation medium for a-galactosidase production by Aspergillus niger zju-Y1was composed of soybean meal1.3782g, wheat bran8.0916g, guar gum0.4802g, KH2PO40.01g, MnSO4·H2O0.04g, the a-galactosidase activity reached265.934U/g dry matter after96h incubation under the optimized medium.Aspergillus niger zju-Yl had shown good ability of enzyme production like cellulase, a-amylase, pectinase and acid proteinase. On the basis of a-galactosidase optimized medium, function model of medium composition and enzyme activity response about cellulase, α-amylase, pectinase and acid proteinase were set up respectively.The ratio of the multi-enzymes was designed. The ingredients of medium were conincided with the design by numerical calculation with method of exhaustion. The multi-enzymes symbolized by a-galactosidase were obtained with Aspergillus niger zju-Y1SSF. The poultry-multi-enzyme A was composed of a-galactosidase243.67U/g dry matter, cellulose431.62U/g dry matter, a-amylase339.77U/g dry matter, pectinase382.19U/g dry matter, acid proteinase3689.16U/g dry matter; The pig-multi-enzyme B was composed of α-galactosidase243.67U/g dry matter, cellulose431.62U/g dry matter, α-amylase339.77U/g dry matter, pectinase382.19U/g dry matter, acid proteinase3689.16U/g dry matter;Each enzyme in multi-enzyme had shown high enzyme activity and good stability in the temperature range at40℃-60℃, and pH3.0-7.0. The multi-enzymes were evaluated with in vitro digestion simulation test. It had shown that the multi-enzymes could improve the digestibility of the feeds effectively. Compared with α-galactosidase, multi-enzymes were more effective on degradation of oligosaccharide.